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Development and Fate of Experimentally Induced Enteritis
GASTROENTE ROLOGY
Vol. 51, No. 5, Part 2 Printed in U .S.A.
Copyright © 1966 by The Williams & Wilkins Co.
DEVELOPMENT AND FATE OF EXPERIMENTALLY INDUCED ENTERITIS JOHN PROHASKA,
M.D.
Department of SU?·gery, University of Chicago, Chicago, Illinois
For many years, investigators have attempted to induce enteritis or colitis in the experimental animal. In some experiments, attempts were made to produce enteritis by the oral administration of fine sand or ground glass. These were crude, unbiological procedures reflecting only physical trauma to the gastrointestinal tract. In the past decade, enteritis and colitis have been induced by the mechanism of the Shwartzman phenomenon. The microbiologists, under the leadership of Gail Dack,l have long been interested in food poisoning. They established the fact that enterotoxin elaborated in the medium of spoiled food by enterotoxigenic strains of StaphylococC1lS var. aureus, produces, in man, debilitating diarrhea, nausea, and vomiting when such food is ingested. The enteritis that ensues is almost never fatal. In the period between 1950 and 1958, a new postoperative complication was observed with alarming frequency and mortality. This complication was marked by green watery diarrhea, abdominal distention, loss of body water and electrolytes, shock, and death. This type of enterocolitis was noted to be associated with the presence of staphylococci in the stools of the afflicted patients as early as 1948. 2 The term "pseudomembranous" enterocolitis was first noted in the literature in an article by Penner and Bernheim, 3 who explained the enterocolitis as a manifestation secondary to shock. Pseudomembranous enterocolitis was not a new disease. It was noted in 1873 by Finney,4 who observed a fatal case following
an operation for peptic ulcer. The disease was noted by several investigators at the beginning of the century. It was referred to as diphtheroid or necrotizing enteritis. It was rare and often confused with enteritis found in heavy metal poisoning. As early as 1902, Riedel 5 referred to diphtheritic membrane in the intestine in 5 fatal cases of enteritis. He was a keen observer and noted the resemblance of diphtheritic enteritis to enteritis caused by heavy metal poisoning. He stated: "This membrane is found notably in the colons of individuals with mercury poisoning; I myself observed necrosis with beginning perforation of the transverse colon following injections of small doses of mercuric oxide." Upon analyzing his 5 fatal cases, he called attention to the possibility that catgut sterilized in alcohol with small additions of mercuric chloride might be the causal agent. He soon discovered that the enteritis occurred in places far away from the suture line in the intestine and abandoned the idea of implicating mercuric chloride. He concluded that the diphtheritic enteritis was the result of poor nutrition. In a survey of 500 pelvic operations, Wertheim 6 lists 3 cases of fatal enteritis. The postmortem findin gs appear to be compatible with pseudomembranous enterocolitis. Diphtheritic membrane in the colon was found in all three cases. Rossle 7 observed, as early as 1912, that the development of certain of these postoperative diarrheas was induced by strong medicated enemas. This, then, was a very important observation since, in the light of our present day knowledge, there is strong evidence th at Staphylococcus does not establish itself readily in the colon unless the normal colonic flora is markedly disturbed. However, Rossie did not associate postoperative diarrheas with staphylococcal in-
Address request for reprints to: Dr. John Prohaska, Professor of Surgery, University of Chicago, 950 E. 59th Street, Chicago, Illinois 60637. This work was supported by Public Health Service Research Grant 1-S01-04-5 from the National Institutes of H ealth. 913
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PROHASKA
vasion of the colon. He attributed the action to the presence of glycerine and turpentine, which were commonly used in the preoperative and postoperative enemas. In 1 of his cases, there was an extensive necrotizing diphtheroid proctitis and colitis. The association of pseudomembranous enterocolitis with vascular changes was first mentioned by Bierende. 8 In 1920, he presented an extensive survey of histological studies performed on 7 necropsy specimens. He observed that most of them showed fixed dilation of blood vessels in the intestinal submucosa. Bierende concluded that the changes in the bowel, known as diphtheritic colitis, were due to vasoparalysis. It is difficult to know whether his cases, complicated by peritonitis, would fulfill our present day definition of enterocolitis. In 1939, Penner and Bernheim 3 studied 40 necropsy specimens taken from patients who died of shock and in whom acute ulcerative or diphtheritic enteritis was found. They concluded that vasomotor mechanisms known to occur in shock were responsible for these lesions. Shock was present in all the cases studied. Similar intestinal lesions were observed after the experimental production of shock. In 1948, Penner and Druckerman 9 reiterated the idea that the vasomotor mechanisms acting in shock were responsible for the condition called necrotizing enterocolitis. By 1950, the writings on pseudomembranous enterocolitis were primarily clinical reports, and the occasional speculations as to the cause of the disease became confused. The earlier search for the cause, initiated by the German investigators, seemed to have been buried by the passage of time. Some clinicians were unable to isolate pathogens in the stools of patients suffering with this complication. Others found Staphylococcus aureus routinely. In 1948, Kramer 2 published perhaps the first report of isolation of Staphylococcus aureus in stools of a patient with enteritis developing in the course of oral streptomycin therapy. I n a series of cases reported by Dixon and vVeismann,10 no pathogen was found which they could incriminate as
Vol. 51, No.5, Part 2
the responsible bacterial agent. In a group of patients reported by Prohaska et al.,n only one positive culture of Staphylococcus aureus was obtained in 7 cases. As late as June 1954, Pettet et al.1 2 wrote: "Unfortunately, after thorough investigation of both the clinical and pathological aspects of this disease, vve are still in the dark as to the etiological factors involved." Yet, the numerous published reports of this disease showed overwhelming evidence in support of the existence of Staphylococcus aureus, often in pure cultures, in the intestinal tract of patients dying from pseudomembranous enterocolitis. Investigators, such as Brown et al., 13 began to find a connection between staphylococcal enteritis and antibiotic therapy. Fairlie and Kendall 14 reported fatal "staphylococcus enteritis" after penicillin and streptomycin therapy. A relatively large series of cases of fulminating gastroenterocolitis was reported by Terplan et aP 5 In this series of 8 cases, all showed pure cultures· of Staphylococcus aureus in the stools. The evidence that staphylococci were implicated in pseudomembranous enterocolitis was observed, not only by the American investigators, but also by the British writers, exemplified by Kramer 2 and Gardner,l 6 who reported in Lancet in 1948 and 1953, respectively. In 1954, Rentchnick 17 published an extensive treatise on the complications of antibiotics in which staphylococcal enterocolitis was frequently mentioned. In most of the cases reported by Rentchnick, pure cultures of staphylococci were obtained from the stools of patients suffering with the disease. The acceptance of Staphylococcus aureus as the logical agent of pseudomembranous enterocolitis was delayed by the reports of Penner and Bernheim, 3 who sought to explain the disease on the basis of histological findings associated with irreversible shock. Their report in 19393 and that of Penner and Druckerman 9 in 1948 gained some support in the combined writings of Hardaway and his associates,l 8 who concluded that pseudomembranous enterocolitis was really the result of two separate phenomena which
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EXPERIMENTALLY INDUCED ENTERITIS
may or may not overlap. They thought that some cases of pseudomembranous enterocolitis could be explained on the basis of intravascular coagulations in the capillaries of the submucosa of the bowel in instances of sudden, severe, refractory shock and in other instances by the disturbance of bacterial flora, in the train of which there develops a staphylococcus enteritis and diarrhea. The idea that pseudomembranous enterocolitis results from vasomotor changes or from intravascular clotting, the result of irreversible shock, is no longer tenable. It is true that sudden, irreversible shock produces vascular changes that are clearly visible in the bowel mucosa. With these changes, areas of intravascular clotting appear in the submucosa. These phenomena are due to sequestration of blood in the vascular venous bed and are observed in shock produced by several endotoxins, as well as in irreversible shock produced in normal animals by the sudden injections of antihypertensive drugs. They are not observed in hemorrhagic shock because the diminished circulatory volume of blood diminishes the amount of pooled blood on the venous side of the capillaries. It is only natural that when a "new" disease is encountered, its cause is at first expressed in terms of the common factors with which the disease may be associated. When multiple associations are observed, as in pseudomembranous enterocolitis, each investigator may emphasize a different set of such correlations. It is obvious, therefore, that an author could single out any one of the factors associated with enterocolitis and place an undue emphasis upon it. In the development of our knowledge relative to enterotoxin enterocolitis, factors like surgical trauma, shock, intravascular clotting, and several microorganisms have been singled out as the possible etiological agents of the disease. These events are almost identical to those presently taking place in the search for etiological agents of ulcerative colitis, in which instance, psychogenic, lysozymal, allergic, and autoimmune factors are offered with equal force and conviction.
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In the period between 1950 and 1956, many surgical patients, during the early postoperative interval, developed green stool diarrhea, abdominal pain with distention, circulatory collapse, and death. The postmortem examination revealed severe enteritis with pseudomembrane under which pure cultures of Staphylococcus aureus could be isolated. At about this time, it was observed that chinchillas receiving chlortetracycline (Aureomycin) pellets in their diets to improve their fur, died from shocklike conditions associated with bloody diarrhea.19 At necropsy, these animals revealed typical enterocolitis with pure cultures of staphylococci in the intestinal lumen. At the same time, it was known that the common laboratory animals, such as the dog, rabbit, guinea pig, and others, were resistant to Staphylococcus and, thus, unsuitable for experiments involving intraluminal survival of staphylococci. With this background, the following experiments were performed. Experimental Observations
In the first series of experiments, 20 chinchillas were used. Twehie animals were experimented upon, and 8 were used as a control. All the experimental chinchillas received an oral administration of an antibiotic mixture consisting of 12.5 mg of chlortetracycline (Aureomycin), 12.5 mg of oxytetracycline (Terramycin), and 25 mg of sulfisoxazole (Gastrisin) daily for 6 to 21 clays. Beginning on clay 6, 2 ml of a normal saline suspension of a pure culture of Staphylococcus attreus was given orally once or twice daily until death occurred. All experimental animals developed lethargy, diarrhea, anorexia, and died in shocklike condition. Severe enterocolitis was found at necropsy, and pure cultures of Staphylococcus aureus were reisolated from their intestines. This reisolatecl microorganism again produced enterotoxin, giving strong positive enterotoxin reaction in the monkey. Gross and microscopic sections showed severe enterocolitis with typical pseudomembrane (fig. 1). The strains of staphylococci used in these experiments were isolated from stools
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Fm. 1. Suspension of a pure culture of Staphylococcus aureus, isolated , from human pseudomembranous enterocolitis, induces in antibiotically prepared chinchilla severe and fatal enteritis. A, Normal upper jejunum. B, J ejunum of an experimental chinchilla showing destruction of mucosa, edema of bowel wall and pseudomembrane (X 100) ~- (Reprinted by permission from A. M. A. Arch. Surg. 79: 197-206, 1959.) -
of patients seriously ill with pseudomembranous enteritis. They were isolated in pure cultures in every case and tested for enterotoxin by the kitten or monkey tests. 20 One specific isolate, for which a precipitation reaction was known, was assayed by the gel precipitant test of Oudin. 21 Every isolate of Staphylococcus aureus used was capable of elaborating potent enterotoxin in vitro. Two to 5 ml of the extract given to a kitten or monkey produced violent retching and diarrhea. A great deal of attention was given to the primary isolation of staphylococci from the stools of patients. Staphylococcus aureus demands a culture medium with strict nutritive requirements. Blood-agar medium is satisfactory and was used most frequently. In other instances, the primary
isolates were grown in the phenylethyl-alcohol (PE) medium of Brewer _and Lilley. For stools, suspected of mixed microbial content, the highly selective tellurite-glycine medium of Zebovitz_ et al. 22_ was employed. One obvious control had ' to be . established, that of an oral administration of the antibiotic mixture alone. For this purpose, 8 normal chinchillas were ·selected. They were given the antibiotic · mixtur-e, previously described, for a pefiod of 10 days. They were observed for -30 ·days. During this period, they remained healthy and free of symptoms. One additionaJ normal, untreated chinchilla was sacrificed in order to obtain normal specimens of the gastrointestinal tract for a comparison,_ with that of the experimental animal.
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These experiments implicate Staphylococcus aureus as the etiological agent of experimentally induced enterocolitis. Under ordinary circumstances, the search for further understanding of the disease might have been terminated. In the instance of Staphylococcus aureus, it was known that the microorganism elaborates enterotoxin. It, therefore, became mandatory to devise experiments in which enterotoxin alone would be employed as the inciting agent. Cats and kittens were known to be sensitive to the enterotoxin and, therefore, were selected for the experiment. It had been established in previous experiments that antibiotic-treated chinchillas were able to grow Staphylococcus aureus in their intestines. If the symptoms of the enteritis and the death of the animals were due to the enterotoxin elaborated in vivo, then the same events should be reproducible in normal animals by the oral administration of the enterotoxin prepared in vitro. Thus, chinchillas had to be included in the enterotoxin experiments.
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Six normal cats received daily or twice daily, 10 ml of fresh enterotoxin by mouth for a period of 12 to 34 days. They all developed symptoms of enteritis, marked by lethargy, anorexia, and diarrhea. They were sacrificed on the 12th to 34th day. At necropsy, all the cats showed gross evidence of enteritis, usually limited to the proximal jejunum. The microscopic sections through the involved jejunum showed, in each instance, a marked leukocytic infiltration and denudation of the mucosa, with sloughing of the villi, erosions of the muscularis, and submucosal edema. Periodic cultures of the stools showed no evidence of staphylococci at any time. The enteritis was induced entirely by the enterotoxin (fig. 2). In the second assignment of the experiment, 7 kittens were given a daily dose of 10 ml of enterotoxin orally for 12 to 23 days. Their symptoms of enteritis, as well as the gross and microscopic findings at necropsy, were found to be more severe than those observed in the cats (figs. 3 and 4).
Fw. 2. Enteritis induced in cat by oral administration of staphylococcal exotoxin. Segment of jejunum showing disruption of intestinal villi, superficial ulcerations, ancl edema of bowel wall (X 42). Reprinted by permission from Prohaska et al.,20 Surg. Gynec: Obstet.109: 73-77, 1959.)
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FIG. 3. Enteritis induced in the kitten by oral administration of staphylococcal exotoxin. A, Normal jejunal segment in a control animal. B, Segment of jejunum in an experimental animal, showing hyperemia , edema, and hemorrhagic mucosa (X 2). (Reprinted by permission from A.M. A. Arch. Surg. 79: 197-206, 1959.)
FIG. 4. Staphylococcal exotoxin enteritis in a kitten. The microscopic section shows destruction of mucosa, minute ulcerations, and round cell infiltration (low power). (Re- . printed by permission from Prohaska et al.,"" Surg. Gynec. Obstet. 109: 73-77, 1959.) ·:,.
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In the terminal phase of the experiment, 20 chinchillas were placed on a daily oral intake of 4 ml of enterotoxin until death occurred in 3 to 12 days. Within a few hours following the administration of the first close of enterotoxin, the chinchillas developed severe diarrhea, lethargy, and tenesmus. Wit h each subsequent daily dose, t hey became weaker, and they died in a convulsive state. At necropsy, t he gross findin gs were identical with those found in the antibiotic-t reated chinchillas receivin g the staphylococcal suspension. The en tire gastrointestinal tract was distended and edematous. The small bowel showed hemorrhagic mucosa. The normal segmentations in the colon were absent and the lumen filled with watery stool. The microscopic section of the stomach, duocle-
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num, jejunum, and colon showed destruction of the mucosa, submucosal edema, deep ulcerations, and fibrinomucous pseudomembrane. A typical section, that of the duodenum, is illustrated in figure 5. Could this disease be reproduced in the clog, an ideal experiment al animal? It was stated that the usu al experimental animal, particularly the clog, was highly resistant to S taphylococcus aureus. The chinchilla and kitten experiments supported the idea t hat enterocolitis occurred in man because the removal of t he normal intestinal flor a produced a condition favorable to the growth of enterotoxigenic staphylococci able to elaborate exotoxin within the 1mman intestine. Therefore, a direct intraluminal administration of the exotoxin appeared to be the cruci al test. This was
Fw. 5. Severe duodenitis induced in the normal, untreated chinchilla by oral administration of staphyloccal exotoxin. Note the edematous thickening of the bowel wall, destruction of mucosa, and formation of debris producing a pseudomembrane (X 110.) (Reprinted by permission from Prohaska et a!.,"' Surg. Gynec. Obstet. 109: 73-77, 1959.)
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accomplished by a Roux-en-Y enterocutaneous fistula, which made it possible to infuse a measured quantity of staphylococcal enterotoxin directly into the midjejunum of the dog. In the course of the study of the biological action of staphylococcal exotoxin, it was observed that a single intraluminal infusion of the toxin induced in the dog severe, acute enteritis (fig. 6). Repeated, daily intraintestinal infusions induced chronic enteritis (fig. 7). The reaction of the intestinal mucosa is constant, quantitative, and reproducible in all experimental dogs. It has been reproduced in 100 canine experiments. Such a constant phenomenon gives rise to some obvious questions. What reaction may be provoked by an intravenous infusion of the staphylococcal exotoxin? If hemodynamic changes should occur, would they be similar to those induced by endotoxins extracted from gram-negative microorganisms? The staphylococcal exotoxin is a mixture of several enzymes and toxins, one of which is enterotoxin. Therefore, our second curiosity was whether the hemodynamic changes, if induced, were caused by the pure enterotoxin or by the entire staphylococcal exotoxin complex. Two preparations were obtained: (I) staphylococcal exotoxin from staphylococcus S-6, an enterotoxigenic strain, and (2) lyophilized pure enterotoxin from the same organism. Thirty-four adult mongrel dogs were used in the study. Under sodium pentabarbitol anesthesia, polyethylene catheters were inserted through the right femoral artery into the abdominal aorta, through the femoral vein into the inferior vena cava, and through a small branch of the splenic vein into the portal vein. The pressure recordings were obtained with straingages on a direct writing polygraph. In some animals, the lymph outflow was measured through a canula placed into the terminal portion of the thoracic duct. The following results were obtained: 1. Staphylococcal exotoxin, administered intravenously, causes an immediate systemic arterial hypotension, portal hypertension, and death of the animal, few
Vol. 51, No . 5, Part Z
minutes following the injection of a minimal lethal dose (fig. 8). During the interval of portal hypertension, the thoracic duct outflow is 6 times normal values, and the lymph often is bloody. 2. Staphylococcal exotoxin administered intraintestinally induces severe enteritis and a slow, gradual lowering of the systemic arterial pressure with a slow rise of portal pressure and death of the animal in 16 hr (fig. 9). 3. Intraluminal administration of pure staphylococcal enterotoxin induces severe enteritis without significant hemodynamic changes in 9 hr. Pure staphylo coccal enterotoxin administered intravenously does not induce significant systemic arterial hypotension, nor a significant rise in portal pressure, nor death of the animal (fig. 10). The purified staphylococcal enterotoxin, whether introduced intravenously or intraintestinally, induces severe enteritis (fig. 11). The enteritis provoked by an intravenous injection of the purified staphylococcal enterotoxin has been reproducible in every dog thus far studied. In some experimental dogs, the enteritis is associated with mild hemorrhagic pancreatitis. This reaction is not predictable. Its nature has not been studied. Conclusions
Staphylococcal crude exotoxin injected intravenously induces arterial hypotension, portal hypertension, increase lymph outflow from the thoracic duct, and death of the animal from circulatory collapse. Staphylococcal purified enterotoxin injected intravenously does not induce circulatory collapse of the animal, but evokes a severe enteritis associated, in some cases, with mild hemorrhagic pancreatitis. Staphylococcal enterotoxin, whether administered intraintestinally or intravenously, evokes a severe enteritis. The response of the intestinal mucosa to staphylococcal exotoxin and staphylococcal enterotoxin is constant, quantitative, and reproducible. Single intraintestinal infusion produces acute enteritis, repeated
.Yocember 1966
EXPERIMENTALLY INDUCED E NTERI1'I S "
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FIG . 6. Dog's jejunum ( X 250) . An acute response to intraintestinally infused staphylococcal exotoxin. The low power microscopic section shows hyperemia, edema, inflammation, d estruction of villi, and ulcerations. (Reprinted by permission from Prohaska et al.,"" Surg. Gyner. Obstet.109: 73-77, 1959.)
FIG. 7. Repeated daily intraintestinal infusions of staphylococcal exotoxin induce in the dog chronic enteritis with lymphoid hyperplasia. A, E arly lymphoid hyperplasia with fragmentation of villi (X 200). B, Advanced evolution of chronic enteritis with marked lymphoid hyperplasia and atrophy of the mucosa ( X 250).
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Vol . 51, N o. 5, Pa rt 2
PROHASKA
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FIG. 8. An intravenous inj ection of crude staphylococcal exotoxin in a dose of 2 ml per kg produces in the dog, rapid rise of portal pressure and an arterial hypotension. The animal dies in 4 min. e, Arterial pressure in millimet ers of Hg; • , venous pressure in millimet ers of H.O ; A, portal pressure in millimeters of H,O. (R eprinted by permission from J. Surg. R es. 6: No.2, 1960.) (MAYDL FisTULA): 30 ML/ KG/ BODY WE IGH T
INFUSION
FIG. 9. An intraintestinal administration of crude staphylococcal exotoxin in a dose of 30 ml per kg induces, in the dog, a slow rise in portal pressure to a peak in 4 hr at which time it declines slowly with a drop in the arterial pressure and a rise in venous pressure. The animal dies in 16 hr. •, Arterial pressure in millimeters of Hg; •· venous pressure in millimeters of H.O; A, portal pressure in millimet ers of H.O. (Reprinted by permission from J. Surg. Res. 6: No.2, 1960.)
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EXPERIMENTALLY INDUCED ENTERITIS
923
Fro. 10. An intravenous injection of purified staphylococcal enterotoxin in a dose of 0.3 mg per kg induces a slight, transient elevation of the portal pressure, without measurable ch anges in the arterial pressure. The inj ection is not lethal. •, Arterial pressure in millimeters of Hg ; II, venous pressure in millimeters of H,O; .A, portal pressure in millimeters of H,O. (Reprinted by permission from J. Surg. Res. 6: No.2, 1960.)
infusions induce chronic enteritis, and large doses are lethal. The fate of such experimentally induced enteritis rests on the exploitation of the experimental model described. It rests on the appreciation of the similarities that exist between the hemodynamic events induced by the gram-negative endotoxins and those induced by the staphylococcal exotoxins. Finally, it rests on the ability and willingness to systematically explore the possible relationship between the experimentally induced enteritis and the human inflammatory diseases of the bowel. REFERENCES 1. D ack, G. M. 1957. Food poisoning. University
Fro. 11 . An intravenous inj ection of 0.2 mg per kg of purified staphylococcal enterotoxin induces, in the dog, severe enteritis. The microscopic section reveals absence of surface epithelium, diffuse infiltration of the stroma by round cells, and ulcerations (X 175).
of Chicago Press, Chicago. 2. Kramer, I. R. H. 1948. Fatal staphylococcal enteritis developing during streptomycin therapy by mouth. Lancet 2: 646-647. 3. Penner, A., and A. I. Bernheim. 1939. Acute postoperative enterocolitis: A study on the pathologic nature of shock. Arch. Path. 27 : 966-983 . . 4. Finney, J . M. T. 1893. Gastroenterostomy for cicatrizing ulcer of pylorus. Bull. H opkins Hosp. 4: 53-55. 5. Riedel, J, 1902. Ueber darmdiphtherie nach schweren operationen bei sehr geschwachten kranken. Deutsch. Z. Chir. 67: 402-413.
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DISCUSSION
6. Wertheim, E. 1911. Operation bei carcinom
colli uteri. In Die erweiterte Abdominale. Urban and Schwarzenberg, Berlin. 7. Rossie, R. 1912. Munchener gynakologische gesellschaft, sitzung vom 16 Marz 1911. Mschr. Geburtsh. Gynac. 35: 243-245. 8. Bierende, F. 1920. Ueber postoperative proct itis und colitis. Mitt. Grenzgeb. Med. Chir. 32: 85-93. 9. P enner, A., and L. J . Druckerman. 1948. Enterocolitis as a postoperative complication and its significance. Gastroenterology 11: 478-487. 10. Dixon, C. F., and R. C. Weismann. 1948. Acute
pseudomembranous enteritis or enterocolitis; complication following intestinal surgery. Surg. Clin. N. Amer. 28: 999-1023. 11. Prohaska, J. V., M. C. Govostis, and M. Taubenhaus. 1954. Postoperative pseudomembranous enterocolitis; successful treatment with corticotropin (ACTH). J. A. M.A. 154: 320-323. 12. Pettet, J . D., A. H. Baggenstoss, E. S. Judd, Jr., and vV. H. Dearing. 1954. Generalized
postoperative pseudomembranous enterocolitis. Mayo Clin. Proc. 29: 342-349. 13. Brown, vV. J., R. Winston, and S. C. Sommers. 1953. Membranous staphylococcal enteritis after antibiotic therapy; report of 2 cases. Amer. J. Dig. Dis. 20 : 73-75. 14. Fairlie, C. W., and R. E. I(endall. 1953. Fatal staphylococcus enteritis following penicillin and streptomycin therapy. J. A. M. A. 153: 90-94. 15. Terplan, K., J. R. Paine, J. Sheffer, R. Egan,
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and H. Lansky. 1953. Fulminating gastroenterocolitis caused by staphylococci: Its apparent connection with antibiotic medication. Gastroenterology 24: 476-509. 16. Gardner, D. L. 1953. Aureomycin-resistant staphylococcus enterocolitis. L ancet 2: 12361238. 17. Rentchnick, P. 1954. Les accidents provoques
par les antibiotiques. Antibiot. Chemother. (Basel) 1: 96-179. 18. Hardaway, R. M., D. G. McKay, C. H. Wahle, and R. N. Hall. 1955. Experimental pseudomembranous enterocolitis: Production by means of thrombosis of intestinal mucosal capillaries. A. M . A. Arch. Intern. Med. 95: 779-787. 19. Bennett, I. L., Jr., J. S. Wood, Jr., and J . H. Yardley. 1956. Staphylococcal pseudomem-
branous enterocolitis in chinchillas: A clinicopathologic study. Trans. Ass. Amer. Physicians 69: 116-121. 20. Prohaska, J. V., M. J. Jacobson, C. T. Drake, and Toh Leong Tan. 1959. Staphylococcus enterotoxin enteritis. Surg. Gynec. Obstet. 109: 73-77. 21. Oudin, J. 1952. Specific precipitation in gels and its application to immunochemical analysis, p. 335-376. In Methods in medical research, Vol. 5. Chicago Year Book Publishers, Inc., Chicago. 22. Zebovitz, E., J. B. Evans, and C. F. Niven, Jr. 1955. Tellurite-glycine agar: A selective plating medium for quantitative detection of coagulase-positive staphylococci. J. Bact. 70: 686-690.
DISCUSSION OF PAPER PRESENTED BY DR. PROHASE:A DR. G. M. DACK (Chicago): It has been interesting to us that most strains that have come from cases of pseudomembranous colitis have been those that produce enterotoxin B. These enterotoxins are getting as complicated as the enterotoxins of Botulinus by developing different antigenic types. I would like to ask Dr. Prohaska if he used only strains isolated from a case of pseudomembranous enterocolitis? DR. PROHASKA: Yes, Dr. Dack. DR. VALDES-DAPENA (Philadelphia) : This interesting study has achieved something that has been lacking in most of the other attempts to reproduce enteritis. Dr. Fahrlander, in Switzerland, has produced
a number of granulomatous changes in the submucosa and the mesentery. He vvas unable to produce any mucosal lesion. This may not be the universal opinion, but I believe chronic enteritis begins in the mucosa. I do not believe that it begins in the mesentery or lymphatics, in all cases. Some combination between this approach of producing the acute injury in the mucosa and sensitization of the animal may lead to a chronic state of enteritis. DR. PROHASKA: vVe have been able to maintain a chronic enteritis with very marked lymphoid hyperplasia and skip areas not dissimilar to those found in regional enteritis by daily intraintestinal in-
November 1966
DISCUSSION
fusion of the whole staphylococcal extract complex for as long as 150 days. However, once you stop the provoking agent healing occurs, and the dog returns to normal. In regard to sensitization, I can state that if you inject the exotoxin intraluminally so that the dog develops a severe diarrhea but does not die, intravenous exotoxin several days later produces death almost instantaneously with a more severe enteritis histologically. DR. HELMUTH SPRINZ (Washington, D. C.): Dr. Kent of my department has just completed a study on the oral administration of staphylococcal enterotoxin B, as a purified compound. vVe were impressed by the rapid development of lesions, within a matter of hours. 'Ve noted changes not only in the stomach, but also in the upper small inte:otine. Our interest in this work wa:o stimulated by the finding of my former a:osociate, Dr. Oscar Felsenfeld, that, in Thailand, the staphylococcal enterotoxin \\·a::; present in the food on the open market and that indigenous people had antibody to staphylococcal enterotoxin in their serum (Proc. Congr. Indian Microbial. Ass. 1: 28, 1959). Since the poor people in Thailand are known to have a spruelike pattern of their intestinal mucosa, we were interested in associating these two findings. Europeans living in Southeast Asia develop similar changes which revert to normal upon leaving this area (J. Trop. Med. Hyg. 67: 300, 1964). DR. DAcre The diseased colons in patients with ulcerative colitis are subject to remi:osions and exacerbations of the disease process when isolated from the rest of the bowel following ileostomy. When the disease process in the isolated colon is active, a Bacteroides jundulijormis (Bacterium necrophorwn) predominates. Antibodies are found in the serum of such patients to the specific strains of B. fundulifonnis found in the bowel but not to other organisms, such as Escherichia coli. B. jundulijormis is found in the ulcerated colon of patients ·with ulcerative colitis who have not had ileostomies. B . junduliformis is found in ulcerated mucous surfaces in other parts of the body as well as the colon, and its role in the etiology of ulcerative colitis
925
is unknown. The question of whether or not ulcerative colitis could develop in the absence of B. jundulijonnis remains unanswered. As knowledge is gained of the cause of ulcerative colitis through studies involving many disciplines, it is hoped that the role of B. fundulijormis in this disease will be explained. DR. NoRMAN ZAMCHECK (Boston): Most of us who have worked with acute inflammatory disorders of the intestine have been impressed with the essentially nonspecific nature of the lesion. I vvonder if Dr. Prohaska or Dr. Sprinz might comment on this. DR. PROHASKA: I agree that the intestine responds in the same fashion with an enteritis or colitis regardless of the type of provoking agent. I have no explanation for this, but the pathologists have known this for a long time. DR. CHARLES H. BROWN (Cleveland): Have ACTH or steroids been of any value in preventing the death or ameliorating the disease of staphylococcal enteritis in animals? DR. PROHASKA: It has not been possible to quantitate the beneficial effect of ACTH or steroids on the severity of the disease or on the survival of the animals subjected to measured do:oes of staphylococcal exotoxin. The evaluation of every harvested exotoxin depends on giving enough of the toxin until symptoms develop or death occurs. DR. BROWN: vVhat are your own thoughts on the etiology of regional enteritis? DR. PROHASKA: The etiology of regional enteritis is unknown. My personal feeling is that exotoxins or endotoxins or some other bacterial products have not been investigated as possible causes of regional enteritis. The experimental enteritis produced by staphylococcal enterotoxin certainly resembles human regional enteritis, grossly and microscopically. The experimental enteritis, like regional enteritis, often appears in skip areas and is associated with lymph node hypertrophy. I do not mean to advance this as the cause of regional enteritis, but I do feel that bacterial products, such as endotoxins, should be investigated.
Vol. 51, No. 5, Part 2 Printed in U .S.A.
Copyright © 1966 by The Williams & Wilkins Co.
DEVELOPMENT AND FATE OF EXPERIMENTALLY INDUCED ENTERITIS JOHN PROHASKA,
M.D.
Department of SU?·gery, University of Chicago, Chicago, Illinois
For many years, investigators have attempted to induce enteritis or colitis in the experimental animal. In some experiments, attempts were made to produce enteritis by the oral administration of fine sand or ground glass. These were crude, unbiological procedures reflecting only physical trauma to the gastrointestinal tract. In the past decade, enteritis and colitis have been induced by the mechanism of the Shwartzman phenomenon. The microbiologists, under the leadership of Gail Dack,l have long been interested in food poisoning. They established the fact that enterotoxin elaborated in the medium of spoiled food by enterotoxigenic strains of StaphylococC1lS var. aureus, produces, in man, debilitating diarrhea, nausea, and vomiting when such food is ingested. The enteritis that ensues is almost never fatal. In the period between 1950 and 1958, a new postoperative complication was observed with alarming frequency and mortality. This complication was marked by green watery diarrhea, abdominal distention, loss of body water and electrolytes, shock, and death. This type of enterocolitis was noted to be associated with the presence of staphylococci in the stools of the afflicted patients as early as 1948. 2 The term "pseudomembranous" enterocolitis was first noted in the literature in an article by Penner and Bernheim, 3 who explained the enterocolitis as a manifestation secondary to shock. Pseudomembranous enterocolitis was not a new disease. It was noted in 1873 by Finney,4 who observed a fatal case following
an operation for peptic ulcer. The disease was noted by several investigators at the beginning of the century. It was referred to as diphtheroid or necrotizing enteritis. It was rare and often confused with enteritis found in heavy metal poisoning. As early as 1902, Riedel 5 referred to diphtheritic membrane in the intestine in 5 fatal cases of enteritis. He was a keen observer and noted the resemblance of diphtheritic enteritis to enteritis caused by heavy metal poisoning. He stated: "This membrane is found notably in the colons of individuals with mercury poisoning; I myself observed necrosis with beginning perforation of the transverse colon following injections of small doses of mercuric oxide." Upon analyzing his 5 fatal cases, he called attention to the possibility that catgut sterilized in alcohol with small additions of mercuric chloride might be the causal agent. He soon discovered that the enteritis occurred in places far away from the suture line in the intestine and abandoned the idea of implicating mercuric chloride. He concluded that the diphtheritic enteritis was the result of poor nutrition. In a survey of 500 pelvic operations, Wertheim 6 lists 3 cases of fatal enteritis. The postmortem findin gs appear to be compatible with pseudomembranous enterocolitis. Diphtheritic membrane in the colon was found in all three cases. Rossle 7 observed, as early as 1912, that the development of certain of these postoperative diarrheas was induced by strong medicated enemas. This, then, was a very important observation since, in the light of our present day knowledge, there is strong evidence th at Staphylococcus does not establish itself readily in the colon unless the normal colonic flora is markedly disturbed. However, Rossie did not associate postoperative diarrheas with staphylococcal in-
Address request for reprints to: Dr. John Prohaska, Professor of Surgery, University of Chicago, 950 E. 59th Street, Chicago, Illinois 60637. This work was supported by Public Health Service Research Grant 1-S01-04-5 from the National Institutes of H ealth. 913
914
PROHASKA
vasion of the colon. He attributed the action to the presence of glycerine and turpentine, which were commonly used in the preoperative and postoperative enemas. In 1 of his cases, there was an extensive necrotizing diphtheroid proctitis and colitis. The association of pseudomembranous enterocolitis with vascular changes was first mentioned by Bierende. 8 In 1920, he presented an extensive survey of histological studies performed on 7 necropsy specimens. He observed that most of them showed fixed dilation of blood vessels in the intestinal submucosa. Bierende concluded that the changes in the bowel, known as diphtheritic colitis, were due to vasoparalysis. It is difficult to know whether his cases, complicated by peritonitis, would fulfill our present day definition of enterocolitis. In 1939, Penner and Bernheim 3 studied 40 necropsy specimens taken from patients who died of shock and in whom acute ulcerative or diphtheritic enteritis was found. They concluded that vasomotor mechanisms known to occur in shock were responsible for these lesions. Shock was present in all the cases studied. Similar intestinal lesions were observed after the experimental production of shock. In 1948, Penner and Druckerman 9 reiterated the idea that the vasomotor mechanisms acting in shock were responsible for the condition called necrotizing enterocolitis. By 1950, the writings on pseudomembranous enterocolitis were primarily clinical reports, and the occasional speculations as to the cause of the disease became confused. The earlier search for the cause, initiated by the German investigators, seemed to have been buried by the passage of time. Some clinicians were unable to isolate pathogens in the stools of patients suffering with this complication. Others found Staphylococcus aureus routinely. In 1948, Kramer 2 published perhaps the first report of isolation of Staphylococcus aureus in stools of a patient with enteritis developing in the course of oral streptomycin therapy. I n a series of cases reported by Dixon and vVeismann,10 no pathogen was found which they could incriminate as
Vol. 51, No.5, Part 2
the responsible bacterial agent. In a group of patients reported by Prohaska et al.,n only one positive culture of Staphylococcus aureus was obtained in 7 cases. As late as June 1954, Pettet et al.1 2 wrote: "Unfortunately, after thorough investigation of both the clinical and pathological aspects of this disease, vve are still in the dark as to the etiological factors involved." Yet, the numerous published reports of this disease showed overwhelming evidence in support of the existence of Staphylococcus aureus, often in pure cultures, in the intestinal tract of patients dying from pseudomembranous enterocolitis. Investigators, such as Brown et al., 13 began to find a connection between staphylococcal enteritis and antibiotic therapy. Fairlie and Kendall 14 reported fatal "staphylococcus enteritis" after penicillin and streptomycin therapy. A relatively large series of cases of fulminating gastroenterocolitis was reported by Terplan et aP 5 In this series of 8 cases, all showed pure cultures· of Staphylococcus aureus in the stools. The evidence that staphylococci were implicated in pseudomembranous enterocolitis was observed, not only by the American investigators, but also by the British writers, exemplified by Kramer 2 and Gardner,l 6 who reported in Lancet in 1948 and 1953, respectively. In 1954, Rentchnick 17 published an extensive treatise on the complications of antibiotics in which staphylococcal enterocolitis was frequently mentioned. In most of the cases reported by Rentchnick, pure cultures of staphylococci were obtained from the stools of patients suffering with the disease. The acceptance of Staphylococcus aureus as the logical agent of pseudomembranous enterocolitis was delayed by the reports of Penner and Bernheim, 3 who sought to explain the disease on the basis of histological findings associated with irreversible shock. Their report in 19393 and that of Penner and Druckerman 9 in 1948 gained some support in the combined writings of Hardaway and his associates,l 8 who concluded that pseudomembranous enterocolitis was really the result of two separate phenomena which
November 1966
EXPERIMENTALLY INDUCED ENTERITIS
may or may not overlap. They thought that some cases of pseudomembranous enterocolitis could be explained on the basis of intravascular coagulations in the capillaries of the submucosa of the bowel in instances of sudden, severe, refractory shock and in other instances by the disturbance of bacterial flora, in the train of which there develops a staphylococcus enteritis and diarrhea. The idea that pseudomembranous enterocolitis results from vasomotor changes or from intravascular clotting, the result of irreversible shock, is no longer tenable. It is true that sudden, irreversible shock produces vascular changes that are clearly visible in the bowel mucosa. With these changes, areas of intravascular clotting appear in the submucosa. These phenomena are due to sequestration of blood in the vascular venous bed and are observed in shock produced by several endotoxins, as well as in irreversible shock produced in normal animals by the sudden injections of antihypertensive drugs. They are not observed in hemorrhagic shock because the diminished circulatory volume of blood diminishes the amount of pooled blood on the venous side of the capillaries. It is only natural that when a "new" disease is encountered, its cause is at first expressed in terms of the common factors with which the disease may be associated. When multiple associations are observed, as in pseudomembranous enterocolitis, each investigator may emphasize a different set of such correlations. It is obvious, therefore, that an author could single out any one of the factors associated with enterocolitis and place an undue emphasis upon it. In the development of our knowledge relative to enterotoxin enterocolitis, factors like surgical trauma, shock, intravascular clotting, and several microorganisms have been singled out as the possible etiological agents of the disease. These events are almost identical to those presently taking place in the search for etiological agents of ulcerative colitis, in which instance, psychogenic, lysozymal, allergic, and autoimmune factors are offered with equal force and conviction.
915
In the period between 1950 and 1956, many surgical patients, during the early postoperative interval, developed green stool diarrhea, abdominal pain with distention, circulatory collapse, and death. The postmortem examination revealed severe enteritis with pseudomembrane under which pure cultures of Staphylococcus aureus could be isolated. At about this time, it was observed that chinchillas receiving chlortetracycline (Aureomycin) pellets in their diets to improve their fur, died from shocklike conditions associated with bloody diarrhea.19 At necropsy, these animals revealed typical enterocolitis with pure cultures of staphylococci in the intestinal lumen. At the same time, it was known that the common laboratory animals, such as the dog, rabbit, guinea pig, and others, were resistant to Staphylococcus and, thus, unsuitable for experiments involving intraluminal survival of staphylococci. With this background, the following experiments were performed. Experimental Observations
In the first series of experiments, 20 chinchillas were used. Twehie animals were experimented upon, and 8 were used as a control. All the experimental chinchillas received an oral administration of an antibiotic mixture consisting of 12.5 mg of chlortetracycline (Aureomycin), 12.5 mg of oxytetracycline (Terramycin), and 25 mg of sulfisoxazole (Gastrisin) daily for 6 to 21 clays. Beginning on clay 6, 2 ml of a normal saline suspension of a pure culture of Staphylococcus attreus was given orally once or twice daily until death occurred. All experimental animals developed lethargy, diarrhea, anorexia, and died in shocklike condition. Severe enterocolitis was found at necropsy, and pure cultures of Staphylococcus aureus were reisolated from their intestines. This reisolatecl microorganism again produced enterotoxin, giving strong positive enterotoxin reaction in the monkey. Gross and microscopic sections showed severe enterocolitis with typical pseudomembrane (fig. 1). The strains of staphylococci used in these experiments were isolated from stools
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PROHASKA
Vol. 51, No.5, Part 2
Fm. 1. Suspension of a pure culture of Staphylococcus aureus, isolated , from human pseudomembranous enterocolitis, induces in antibiotically prepared chinchilla severe and fatal enteritis. A, Normal upper jejunum. B, J ejunum of an experimental chinchilla showing destruction of mucosa, edema of bowel wall and pseudomembrane (X 100) ~- (Reprinted by permission from A. M. A. Arch. Surg. 79: 197-206, 1959.) -
of patients seriously ill with pseudomembranous enteritis. They were isolated in pure cultures in every case and tested for enterotoxin by the kitten or monkey tests. 20 One specific isolate, for which a precipitation reaction was known, was assayed by the gel precipitant test of Oudin. 21 Every isolate of Staphylococcus aureus used was capable of elaborating potent enterotoxin in vitro. Two to 5 ml of the extract given to a kitten or monkey produced violent retching and diarrhea. A great deal of attention was given to the primary isolation of staphylococci from the stools of patients. Staphylococcus aureus demands a culture medium with strict nutritive requirements. Blood-agar medium is satisfactory and was used most frequently. In other instances, the primary
isolates were grown in the phenylethyl-alcohol (PE) medium of Brewer _and Lilley. For stools, suspected of mixed microbial content, the highly selective tellurite-glycine medium of Zebovitz_ et al. 22_ was employed. One obvious control had ' to be . established, that of an oral administration of the antibiotic mixture alone. For this purpose, 8 normal chinchillas were ·selected. They were given the antibiotic · mixtur-e, previously described, for a pefiod of 10 days. They were observed for -30 ·days. During this period, they remained healthy and free of symptoms. One additionaJ normal, untreated chinchilla was sacrificed in order to obtain normal specimens of the gastrointestinal tract for a comparison,_ with that of the experimental animal.
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EXPERIMENTALLY INDUCED ENTERITIS
These experiments implicate Staphylococcus aureus as the etiological agent of experimentally induced enterocolitis. Under ordinary circumstances, the search for further understanding of the disease might have been terminated. In the instance of Staphylococcus aureus, it was known that the microorganism elaborates enterotoxin. It, therefore, became mandatory to devise experiments in which enterotoxin alone would be employed as the inciting agent. Cats and kittens were known to be sensitive to the enterotoxin and, therefore, were selected for the experiment. It had been established in previous experiments that antibiotic-treated chinchillas were able to grow Staphylococcus aureus in their intestines. If the symptoms of the enteritis and the death of the animals were due to the enterotoxin elaborated in vivo, then the same events should be reproducible in normal animals by the oral administration of the enterotoxin prepared in vitro. Thus, chinchillas had to be included in the enterotoxin experiments.
917
Six normal cats received daily or twice daily, 10 ml of fresh enterotoxin by mouth for a period of 12 to 34 days. They all developed symptoms of enteritis, marked by lethargy, anorexia, and diarrhea. They were sacrificed on the 12th to 34th day. At necropsy, all the cats showed gross evidence of enteritis, usually limited to the proximal jejunum. The microscopic sections through the involved jejunum showed, in each instance, a marked leukocytic infiltration and denudation of the mucosa, with sloughing of the villi, erosions of the muscularis, and submucosal edema. Periodic cultures of the stools showed no evidence of staphylococci at any time. The enteritis was induced entirely by the enterotoxin (fig. 2). In the second assignment of the experiment, 7 kittens were given a daily dose of 10 ml of enterotoxin orally for 12 to 23 days. Their symptoms of enteritis, as well as the gross and microscopic findings at necropsy, were found to be more severe than those observed in the cats (figs. 3 and 4).
Fw. 2. Enteritis induced in cat by oral administration of staphylococcal exotoxin. Segment of jejunum showing disruption of intestinal villi, superficial ulcerations, ancl edema of bowel wall (X 42). Reprinted by permission from Prohaska et al.,20 Surg. Gynec: Obstet.109: 73-77, 1959.)
918
PROHASIU
1'ol .. 51, No.5, Pat! 2
FIG. 3. Enteritis induced in the kitten by oral administration of staphylococcal exotoxin. A, Normal jejunal segment in a control animal. B, Segment of jejunum in an experimental animal, showing hyperemia , edema, and hemorrhagic mucosa (X 2). (Reprinted by permission from A.M. A. Arch. Surg. 79: 197-206, 1959.)
FIG. 4. Staphylococcal exotoxin enteritis in a kitten. The microscopic section shows destruction of mucosa, minute ulcerations, and round cell infiltration (low power). (Re- . printed by permission from Prohaska et al.,"" Surg. Gynec. Obstet. 109: 73-77, 1959.) ·:,.
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EXPERIMENTALLY I N DUCED E NTERITIS
In the terminal phase of the experiment, 20 chinchillas were placed on a daily oral intake of 4 ml of enterotoxin until death occurred in 3 to 12 days. Within a few hours following the administration of the first close of enterotoxin, the chinchillas developed severe diarrhea, lethargy, and tenesmus. Wit h each subsequent daily dose, t hey became weaker, and they died in a convulsive state. At necropsy, t he gross findin gs were identical with those found in the antibiotic-t reated chinchillas receivin g the staphylococcal suspension. The en tire gastrointestinal tract was distended and edematous. The small bowel showed hemorrhagic mucosa. The normal segmentations in the colon were absent and the lumen filled with watery stool. The microscopic section of the stomach, duocle-
919
num, jejunum, and colon showed destruction of the mucosa, submucosal edema, deep ulcerations, and fibrinomucous pseudomembrane. A typical section, that of the duodenum, is illustrated in figure 5. Could this disease be reproduced in the clog, an ideal experiment al animal? It was stated that the usu al experimental animal, particularly the clog, was highly resistant to S taphylococcus aureus. The chinchilla and kitten experiments supported the idea t hat enterocolitis occurred in man because the removal of t he normal intestinal flor a produced a condition favorable to the growth of enterotoxigenic staphylococci able to elaborate exotoxin within the 1mman intestine. Therefore, a direct intraluminal administration of the exotoxin appeared to be the cruci al test. This was
Fw. 5. Severe duodenitis induced in the normal, untreated chinchilla by oral administration of staphyloccal exotoxin. Note the edematous thickening of the bowel wall, destruction of mucosa, and formation of debris producing a pseudomembrane (X 110.) (Reprinted by permission from Prohaska et a!.,"' Surg. Gynec. Obstet. 109: 73-77, 1959.)
920
PROHASKA
accomplished by a Roux-en-Y enterocutaneous fistula, which made it possible to infuse a measured quantity of staphylococcal enterotoxin directly into the midjejunum of the dog. In the course of the study of the biological action of staphylococcal exotoxin, it was observed that a single intraluminal infusion of the toxin induced in the dog severe, acute enteritis (fig. 6). Repeated, daily intraintestinal infusions induced chronic enteritis (fig. 7). The reaction of the intestinal mucosa is constant, quantitative, and reproducible in all experimental dogs. It has been reproduced in 100 canine experiments. Such a constant phenomenon gives rise to some obvious questions. What reaction may be provoked by an intravenous infusion of the staphylococcal exotoxin? If hemodynamic changes should occur, would they be similar to those induced by endotoxins extracted from gram-negative microorganisms? The staphylococcal exotoxin is a mixture of several enzymes and toxins, one of which is enterotoxin. Therefore, our second curiosity was whether the hemodynamic changes, if induced, were caused by the pure enterotoxin or by the entire staphylococcal exotoxin complex. Two preparations were obtained: (I) staphylococcal exotoxin from staphylococcus S-6, an enterotoxigenic strain, and (2) lyophilized pure enterotoxin from the same organism. Thirty-four adult mongrel dogs were used in the study. Under sodium pentabarbitol anesthesia, polyethylene catheters were inserted through the right femoral artery into the abdominal aorta, through the femoral vein into the inferior vena cava, and through a small branch of the splenic vein into the portal vein. The pressure recordings were obtained with straingages on a direct writing polygraph. In some animals, the lymph outflow was measured through a canula placed into the terminal portion of the thoracic duct. The following results were obtained: 1. Staphylococcal exotoxin, administered intravenously, causes an immediate systemic arterial hypotension, portal hypertension, and death of the animal, few
Vol. 51, No . 5, Part Z
minutes following the injection of a minimal lethal dose (fig. 8). During the interval of portal hypertension, the thoracic duct outflow is 6 times normal values, and the lymph often is bloody. 2. Staphylococcal exotoxin administered intraintestinally induces severe enteritis and a slow, gradual lowering of the systemic arterial pressure with a slow rise of portal pressure and death of the animal in 16 hr (fig. 9). 3. Intraluminal administration of pure staphylococcal enterotoxin induces severe enteritis without significant hemodynamic changes in 9 hr. Pure staphylo coccal enterotoxin administered intravenously does not induce significant systemic arterial hypotension, nor a significant rise in portal pressure, nor death of the animal (fig. 10). The purified staphylococcal enterotoxin, whether introduced intravenously or intraintestinally, induces severe enteritis (fig. 11). The enteritis provoked by an intravenous injection of the purified staphylococcal enterotoxin has been reproducible in every dog thus far studied. In some experimental dogs, the enteritis is associated with mild hemorrhagic pancreatitis. This reaction is not predictable. Its nature has not been studied. Conclusions
Staphylococcal crude exotoxin injected intravenously induces arterial hypotension, portal hypertension, increase lymph outflow from the thoracic duct, and death of the animal from circulatory collapse. Staphylococcal purified enterotoxin injected intravenously does not induce circulatory collapse of the animal, but evokes a severe enteritis associated, in some cases, with mild hemorrhagic pancreatitis. Staphylococcal enterotoxin, whether administered intraintestinally or intravenously, evokes a severe enteritis. The response of the intestinal mucosa to staphylococcal exotoxin and staphylococcal enterotoxin is constant, quantitative, and reproducible. Single intraintestinal infusion produces acute enteritis, repeated
.Yocember 1966
EXPERIMENTALLY INDUCED E NTERI1'I S "
·. ,.
.
•;·· ~
..
FIG . 6. Dog's jejunum ( X 250) . An acute response to intraintestinally infused staphylococcal exotoxin. The low power microscopic section shows hyperemia, edema, inflammation, d estruction of villi, and ulcerations. (Reprinted by permission from Prohaska et al.,"" Surg. Gyner. Obstet.109: 73-77, 1959.)
FIG. 7. Repeated daily intraintestinal infusions of staphylococcal exotoxin induce in the dog chronic enteritis with lymphoid hyperplasia. A, E arly lymphoid hyperplasia with fragmentation of villi (X 200). B, Advanced evolution of chronic enteritis with marked lymphoid hyperplasia and atrophy of the mucosa ( X 250).
921
Vol . 51, N o. 5, Pa rt 2
PROHASKA
922 .TIME IN MINUTES I
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INJECTION TIME
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FIG. 8. An intravenous inj ection of crude staphylococcal exotoxin in a dose of 2 ml per kg produces in the dog, rapid rise of portal pressure and an arterial hypotension. The animal dies in 4 min. e, Arterial pressure in millimet ers of Hg; • , venous pressure in millimet ers of H.O ; A, portal pressure in millimeters of H,O. (R eprinted by permission from J. Surg. R es. 6: No.2, 1960.) (MAYDL FisTULA): 30 ML/ KG/ BODY WE IGH T
INFUSION
FIG. 9. An intraintestinal administration of crude staphylococcal exotoxin in a dose of 30 ml per kg induces, in the dog, a slow rise in portal pressure to a peak in 4 hr at which time it declines slowly with a drop in the arterial pressure and a rise in venous pressure. The animal dies in 16 hr. •, Arterial pressure in millimeters of Hg; •· venous pressure in millimeters of H.O; A, portal pressure in millimet ers of H.O. (Reprinted by permission from J. Surg. Res. 6: No.2, 1960.)
November 1966
EXPERIMENTALLY INDUCED ENTERITIS
923
Fro. 10. An intravenous injection of purified staphylococcal enterotoxin in a dose of 0.3 mg per kg induces a slight, transient elevation of the portal pressure, without measurable ch anges in the arterial pressure. The inj ection is not lethal. •, Arterial pressure in millimeters of Hg ; II, venous pressure in millimeters of H,O; .A, portal pressure in millimeters of H,O. (Reprinted by permission from J. Surg. Res. 6: No.2, 1960.)
infusions induce chronic enteritis, and large doses are lethal. The fate of such experimentally induced enteritis rests on the exploitation of the experimental model described. It rests on the appreciation of the similarities that exist between the hemodynamic events induced by the gram-negative endotoxins and those induced by the staphylococcal exotoxins. Finally, it rests on the ability and willingness to systematically explore the possible relationship between the experimentally induced enteritis and the human inflammatory diseases of the bowel. REFERENCES 1. D ack, G. M. 1957. Food poisoning. University
Fro. 11 . An intravenous inj ection of 0.2 mg per kg of purified staphylococcal enterotoxin induces, in the dog, severe enteritis. The microscopic section reveals absence of surface epithelium, diffuse infiltration of the stroma by round cells, and ulcerations (X 175).
of Chicago Press, Chicago. 2. Kramer, I. R. H. 1948. Fatal staphylococcal enteritis developing during streptomycin therapy by mouth. Lancet 2: 646-647. 3. Penner, A., and A. I. Bernheim. 1939. Acute postoperative enterocolitis: A study on the pathologic nature of shock. Arch. Path. 27 : 966-983 . . 4. Finney, J . M. T. 1893. Gastroenterostomy for cicatrizing ulcer of pylorus. Bull. H opkins Hosp. 4: 53-55. 5. Riedel, J, 1902. Ueber darmdiphtherie nach schweren operationen bei sehr geschwachten kranken. Deutsch. Z. Chir. 67: 402-413.
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DISCUSSION
6. Wertheim, E. 1911. Operation bei carcinom
colli uteri. In Die erweiterte Abdominale. Urban and Schwarzenberg, Berlin. 7. Rossie, R. 1912. Munchener gynakologische gesellschaft, sitzung vom 16 Marz 1911. Mschr. Geburtsh. Gynac. 35: 243-245. 8. Bierende, F. 1920. Ueber postoperative proct itis und colitis. Mitt. Grenzgeb. Med. Chir. 32: 85-93. 9. P enner, A., and L. J . Druckerman. 1948. Enterocolitis as a postoperative complication and its significance. Gastroenterology 11: 478-487. 10. Dixon, C. F., and R. C. Weismann. 1948. Acute
pseudomembranous enteritis or enterocolitis; complication following intestinal surgery. Surg. Clin. N. Amer. 28: 999-1023. 11. Prohaska, J. V., M. C. Govostis, and M. Taubenhaus. 1954. Postoperative pseudomembranous enterocolitis; successful treatment with corticotropin (ACTH). J. A. M.A. 154: 320-323. 12. Pettet, J . D., A. H. Baggenstoss, E. S. Judd, Jr., and vV. H. Dearing. 1954. Generalized
postoperative pseudomembranous enterocolitis. Mayo Clin. Proc. 29: 342-349. 13. Brown, vV. J., R. Winston, and S. C. Sommers. 1953. Membranous staphylococcal enteritis after antibiotic therapy; report of 2 cases. Amer. J. Dig. Dis. 20 : 73-75. 14. Fairlie, C. W., and R. E. I(endall. 1953. Fatal staphylococcus enteritis following penicillin and streptomycin therapy. J. A. M. A. 153: 90-94. 15. Terplan, K., J. R. Paine, J. Sheffer, R. Egan,
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and H. Lansky. 1953. Fulminating gastroenterocolitis caused by staphylococci: Its apparent connection with antibiotic medication. Gastroenterology 24: 476-509. 16. Gardner, D. L. 1953. Aureomycin-resistant staphylococcus enterocolitis. L ancet 2: 12361238. 17. Rentchnick, P. 1954. Les accidents provoques
par les antibiotiques. Antibiot. Chemother. (Basel) 1: 96-179. 18. Hardaway, R. M., D. G. McKay, C. H. Wahle, and R. N. Hall. 1955. Experimental pseudomembranous enterocolitis: Production by means of thrombosis of intestinal mucosal capillaries. A. M . A. Arch. Intern. Med. 95: 779-787. 19. Bennett, I. L., Jr., J. S. Wood, Jr., and J . H. Yardley. 1956. Staphylococcal pseudomem-
branous enterocolitis in chinchillas: A clinicopathologic study. Trans. Ass. Amer. Physicians 69: 116-121. 20. Prohaska, J. V., M. J. Jacobson, C. T. Drake, and Toh Leong Tan. 1959. Staphylococcus enterotoxin enteritis. Surg. Gynec. Obstet. 109: 73-77. 21. Oudin, J. 1952. Specific precipitation in gels and its application to immunochemical analysis, p. 335-376. In Methods in medical research, Vol. 5. Chicago Year Book Publishers, Inc., Chicago. 22. Zebovitz, E., J. B. Evans, and C. F. Niven, Jr. 1955. Tellurite-glycine agar: A selective plating medium for quantitative detection of coagulase-positive staphylococci. J. Bact. 70: 686-690.
DISCUSSION OF PAPER PRESENTED BY DR. PROHASE:A DR. G. M. DACK (Chicago): It has been interesting to us that most strains that have come from cases of pseudomembranous colitis have been those that produce enterotoxin B. These enterotoxins are getting as complicated as the enterotoxins of Botulinus by developing different antigenic types. I would like to ask Dr. Prohaska if he used only strains isolated from a case of pseudomembranous enterocolitis? DR. PROHASKA: Yes, Dr. Dack. DR. VALDES-DAPENA (Philadelphia) : This interesting study has achieved something that has been lacking in most of the other attempts to reproduce enteritis. Dr. Fahrlander, in Switzerland, has produced
a number of granulomatous changes in the submucosa and the mesentery. He vvas unable to produce any mucosal lesion. This may not be the universal opinion, but I believe chronic enteritis begins in the mucosa. I do not believe that it begins in the mesentery or lymphatics, in all cases. Some combination between this approach of producing the acute injury in the mucosa and sensitization of the animal may lead to a chronic state of enteritis. DR. PROHASKA: vVe have been able to maintain a chronic enteritis with very marked lymphoid hyperplasia and skip areas not dissimilar to those found in regional enteritis by daily intraintestinal in-
November 1966
DISCUSSION
fusion of the whole staphylococcal extract complex for as long as 150 days. However, once you stop the provoking agent healing occurs, and the dog returns to normal. In regard to sensitization, I can state that if you inject the exotoxin intraluminally so that the dog develops a severe diarrhea but does not die, intravenous exotoxin several days later produces death almost instantaneously with a more severe enteritis histologically. DR. HELMUTH SPRINZ (Washington, D. C.): Dr. Kent of my department has just completed a study on the oral administration of staphylococcal enterotoxin B, as a purified compound. vVe were impressed by the rapid development of lesions, within a matter of hours. 'Ve noted changes not only in the stomach, but also in the upper small inte:otine. Our interest in this work wa:o stimulated by the finding of my former a:osociate, Dr. Oscar Felsenfeld, that, in Thailand, the staphylococcal enterotoxin \\·a::; present in the food on the open market and that indigenous people had antibody to staphylococcal enterotoxin in their serum (Proc. Congr. Indian Microbial. Ass. 1: 28, 1959). Since the poor people in Thailand are known to have a spruelike pattern of their intestinal mucosa, we were interested in associating these two findings. Europeans living in Southeast Asia develop similar changes which revert to normal upon leaving this area (J. Trop. Med. Hyg. 67: 300, 1964). DR. DAcre The diseased colons in patients with ulcerative colitis are subject to remi:osions and exacerbations of the disease process when isolated from the rest of the bowel following ileostomy. When the disease process in the isolated colon is active, a Bacteroides jundulijormis (Bacterium necrophorwn) predominates. Antibodies are found in the serum of such patients to the specific strains of B. fundulifonnis found in the bowel but not to other organisms, such as Escherichia coli. B. jundulijormis is found in the ulcerated colon of patients ·with ulcerative colitis who have not had ileostomies. B . junduliformis is found in ulcerated mucous surfaces in other parts of the body as well as the colon, and its role in the etiology of ulcerative colitis
925
is unknown. The question of whether or not ulcerative colitis could develop in the absence of B. jundulijonnis remains unanswered. As knowledge is gained of the cause of ulcerative colitis through studies involving many disciplines, it is hoped that the role of B. fundulijormis in this disease will be explained. DR. NoRMAN ZAMCHECK (Boston): Most of us who have worked with acute inflammatory disorders of the intestine have been impressed with the essentially nonspecific nature of the lesion. I vvonder if Dr. Prohaska or Dr. Sprinz might comment on this. DR. PROHASKA: I agree that the intestine responds in the same fashion with an enteritis or colitis regardless of the type of provoking agent. I have no explanation for this, but the pathologists have known this for a long time. DR. CHARLES H. BROWN (Cleveland): Have ACTH or steroids been of any value in preventing the death or ameliorating the disease of staphylococcal enteritis in animals? DR. PROHASKA: It has not been possible to quantitate the beneficial effect of ACTH or steroids on the severity of the disease or on the survival of the animals subjected to measured do:oes of staphylococcal exotoxin. The evaluation of every harvested exotoxin depends on giving enough of the toxin until symptoms develop or death occurs. DR. BROWN: vVhat are your own thoughts on the etiology of regional enteritis? DR. PROHASKA: The etiology of regional enteritis is unknown. My personal feeling is that exotoxins or endotoxins or some other bacterial products have not been investigated as possible causes of regional enteritis. The experimental enteritis produced by staphylococcal enterotoxin certainly resembles human regional enteritis, grossly and microscopically. The experimental enteritis, like regional enteritis, often appears in skip areas and is associated with lymph node hypertrophy. I do not mean to advance this as the cause of regional enteritis, but I do feel that bacterial products, such as endotoxins, should be investigated.